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Vitamin C improves the effect of a new nitric oxide donor on the vascular smooth muscle from renal hypertensive rats
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Nitric Oxide 18 (2008) 176–183 www.elsevier.com/locate/yniox
Vitamin C improves the effect of a new nitric oxide donor on the vascular smooth muscle from renal hypertensive rats G.J. Rodrigues a, C.N. Lunardi a, R.G. Lima a, C.X. Santos b, F.R.M. Laurindo b, R.S. da Silva a, L.M. Bendhack a,* a
Departamento de Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, USP, 14040-903 Ribeira˜o Preto, SP, Brazil b Laborato´rio de Biologia Vascular, Instituto do Coracßa˜o (InCor), USP, 05403-000, Brazil Received 14 September 2007; revised 29 November 2007 Available online 25 December 2007
Abstract Impaired relaxation induced by the new nitric oxide (NO) donor [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) has been observed in the aortic rings from renal hypertensive rats (2K-1C). An increased production of reactive oxygen species (ROS) in the aortas from 2K1C rats are capable of reducing NO bioavailability. Therefore, this study aimed at investigating the effects of an antioxidant (vitamin C) on the relaxant effect of NO released from TERPY on the 2K-1C rat aorta. As for vascular reactivity, the potency of TERPY is greater in the control rats (2K) than in 2K-1C whereas the maximum relaxation (ME) is not significantly different between the 2K and 2K-1C rat aortas. The relaxation of TERPY is potentiated only in the 2K-1C aortic ring treated with vitamin C. TERPY has a lower effect in decreasing cytosolic Ca2+ concentration ([Ca2+]c) in vascular smooth muscle cells (VSMCs) from 2K-1C rats. This effect is also potentiated in 2K-1C aortic cells treated with vitamin C, but it is not altered in 2K cells. The basal cytosolic NO concentration ([NO]c) is lower in 2K-1C than in 2K cells, and the bioavailability of the NO released from TERPY is larger in 2K than in 2K-1C VSMCs. The superoxide radical concentration ([O 2 ]) is higher in the 2K-1C aorta, and vitamin C reduces the [O2 ] in the 2K-1C aorta. Taken together, these results show that in the aortas of renal hypertensive 2K-1C rats, released NO from the new NO donor is not available to produce a similar effect in 2K aorta due to increased [O 2 ]. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Nitric oxide donor; Superoxide anion; Renal hypertension; Antioxidant
Introduction Nitric oxide (NO) regulates an array of physiological processes including the maintenance of vascular tone. The vasorelaxation induced by NO involves mainly the activation of soluble guanylyl cyclase, which catalyzes the conversion of guanosine triphosphate (GTP) into cyclic guanosine monophosphate [1]. Acting as a second messenger, cGMP activates a family of serine/threonine protein kinases called cGMP-dependent protein kinases (PKGs), which induce relaxation by decrease of cytosolic Ca2+ concentration ([Ca2+]c) and Ca2+ desensitization of actin–
*
Corresponding author. Fax: +55 16 36024880. E-mail address: [email protected] (L.M. Bendhack).
1089-8603/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2007.12.002
myosin contractile system, in vascular smooth muscle cells [2]. Various cGMP-independent mechanisms of NO have been reported in a number of studies, including the direct activation of K+ channels [3,4], as well the activation of Na+/K+-ATPase pump [4]. In previous studies, we have observed an impaired relaxation to NO in arteries from renovascular hypertensive rats [5,6]. This impairment has been attributed to the increased production of reactive oxygen species (ROS) [7]. The superoxide (O 2 ) is an important member of the ROS family, which has been found in elevated concentration in the vascular smooth muscle cells from renal hypertensive rats [8– 10]. The NO bioavailability could be significantly reduced in the presence of O 2 because NO reacts with O2 to form peroxynitrite (ONOO ) [11]. These potentially harmful vascular effects of the superoxide are normally counterbal-
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anced by the enzyme superoxide dismutase and other antioxidants such as vitamins E and C [12–16]. An imbalance between the production of ROS and the level of protective antioxidants could lead to significant increases in the oxidative stress and arterial pressure [11]. A restoration of this balance by antioxidant oral treatment with tempol [17,18], sesamin [17,19], vitamin E [19,20], and vitamin C [20] has been shown to decrease the arterial pressure in hypertensive rats. NO donors are pharmacologically active substances that release NO in vivo or in vitro. [Ru(terpy)(bdq)NO+]3+ (TERPY) is a nitrosyl ruthenium complex that is thermally stable in physiological pH and it has been described as an NO donor [21] that induces vascular relaxation. The cellular mechanisms involved in this effect have been described by Bonaventura et al. [22]. This compound is attractive since NO donors used in clinical medicine present some disadvantages. For instance, the chronic treatment with nitroglycerin induces tolerance [23], and sodium nitroprusside releases cyanide [24], which is toxic to the vascular cells. Therefore, the aim of this study was to verify the influence of the antioxidant agent vitamin C on the effect of the new NO donor TERPY on the aortic vascular smooth muscle from renal hypertensive rats (2K-1C). Materials and methods Experimental animals Experimental protocols followed standards and policies of the Animal Care and Use Committee of the University of Sa˜o Paulo. Renovascular hypertension was induced in rats following the 2K-1C Goldblatt model [25]. Briefly, male Wistar rats (180–200 g) were anesthetized with tribromoethanol (2.5 mg kg1, i.p.) and after a midline laparotomy a silver clip with an internal diameter of 0.20 mm was placed around the left renal artery. Normotensive two-kidney rats (2K) were only submitted to laparotomy. Animals were maintained on standard rat chow with a 12 h light/dark cycle and given free access to both food (standard rat chow) and water. The systolic blood pressure (SBP) was measured weekly in non-anesthetized animals by an indirect tail-cuff method (MLT125R pulse transducer/pressure cuff coupled to the PowerLab 4/S analog-to-digital converter; AD Instruments Pty Ltd., Castle Hill, Australia), and rats were considered hypertensive when the SBP was higher than 160 mmHg.
Functional studies Rats were killed by decapitation six weeks after surgery, and the thoracic aortas were isolated. Aortic rings, 4 mm in length, were placed in bath chambers (10 mL) for isolated organs containing physiological salt solution (PSS) at 37 °C, continuously bubbled with 95% O2 and 5% CO2, pH 7.4. Two fine stainless steel holders were placed through the lumen of the aortic rings, one of the holders was fixed to the tissue chamber and the other was connected to an F-60 force–displacement transducer. The contractile/relaxant responses were recorded on a polygraph (Narco Biosystems Inc., Houston, TX, USA). The aortic rings were submitted to a tension of 1.5 g, which was readjusted every 15 min during a 60 min equilibration period before addition of the given drug. An optimal basal tension of 1.5 g in the aortic rings from both 2K and 2K-1C was previously standardized by exposing the vessels to 90 mM KCl under various resting tensions (0.25–2.5 g). The endothelium was mechanically removed by gently rubbing the intimal surface with stainless steel holders. Endothelial integrity was qualitatively assessed by the degree of relaxation caused
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by 1 lM acetylcholine in the presence of contractile tone induced by phenylephrine (0.1 lM). As our studies required endothelium denuded aortas, the rings were discarded if there was any degree of relaxation, to avoid the possible influence of endothelial factors. After endothelial integrity was assessed, aortic rings were pre-contracted with phenylephrine (0.1 lM), and concentration–effect curves to NO donor TERPY (1 nM to 300 lM) were constructed in the 2K and 2K-1C aortic rings when the plateau was reached. Vitamin C (100 lM) [26] was added 20 min before phenylephrine. Concentration–effect curves to TERPY (1 nM to 300 lM) were constructed in order to examine the effect of vitamin C on the relaxation induced by the NO donor, in the aortic rings isolated from 2K and 2K-1C rats pre-contracted with phenylephrine (0.1 lM), in the presence or absence of vitamin C.
Vascular smooth muscle cells isolation Rats were killed by decapitation six weeks after surgery, and the thoracic aortas were isolated. Vascular smooth muscle cells (VSMCs) were isolated by enzymatic digestion from aorta as described previously [29]. Briefly, the aortas were dissected and longitudinally opened. The endothelium and the adventitia were removed, and the tissue was minced into small pieces and incubated in Ca2+ and Mg+ free Hanks solution with the following composition (in mM): 145.0 NaCl, 5.0 KCl, 0.5 NaH2PO4, 10.0 dextrose and 10.0 HEPES (pH 7.4) containing 0.03 mg/mL collagenase Type II-S. The tissue was gently shaken in this solution for 25 min at 37 °C and bubbled with carbogen mixture. After that, 10 mg/mL bovine serum albumin (type I) was added to the vessel fragments in Ca2+ and Mg+ free Hanks solution, and cells were released by mechanical dispersion with a Pasteur pipette. The resultant cell suspension was centrifuged at 1000 rpm for 3 min and suspended in Ca2+ and Mg+ free Hanks. The cells were plated on glass coverslips pretreated with poly-L-lysine solution and kept in a humidified 37 °C incubator gassed with 5% CO2. The cells were used 3 h after plating and were maintained in a serum-free medium. A similar procedure was carried out for 2K and 2K-1C rats.
Ca2+ measurements To confirm the results obtained with TERPY in the aorta tissue by vascular reactivity studies, we used confocal microscopy. In order to assess the transient of the cytosolic Ca2+ concentration ([Ca2+]c), VSMCs were loaded with Fluo 3-AM (10 lM) for 30 min at room temperature. Excess dye was removed by washing out the dye with bath solution and allowing 30 min for intracellular desterification of Fluo 3-AM to Fluo 3. The cells were imaged in Hanks (with the following composition in mM: 1.6 CaCl2, 1.0 MgCl2, 145.0 NaCl, 5.0 KCl, 0.5 NaH2PO4, 10.0 dextrose and 10.0 HEPES, pH 7.4) and [Ca2+]c was assessed by a confocal scanning laser microscope (Leica TCS SP5) [27–30]. Fluo 3 fluorescence was excited with the 488 nm line of an argon ion laser and the emitted fluorescence was measured at 510 nm. A time course software was used to capture images of the cells at intervals of 0.850 s (xyt) in Live Data Mode acquisition. By applying the computer software of the LSCM, the intensities of the intracellular maximum or minimum fluorescence were measured. From this data, it was used as the initial fluorescence intensity value at t = 0 s was taken as F0, and the final value of the fluorescence intensity F was registered after TERPY (10 lM) addition. In this way, negative values from differences in percentage of fluorescence intensity (D%FI) reflects the decrease in [Ca2+]c, which was obtained for each protocol in relation to basal (F0) (100%). Vitamin C (100 lM) was added 20 min before TERPY (10 lM).
D%FI ¼ ðF F 0 =F 0 Þ 100 NO measurements To assess the cytosolic nitric oxide concentration ([NO]c), VSMCs were loaded with the NO indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) (5 lM) for 30 min, at room temperature [31]. The membrane
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permeable DAF-2 DA readily enters the cells and is subsequently hydrolyzed by cytosolic esterases releasing free DAF-2, which does not leak into the medium. At physiological pH, DAF-2 is relatively non-fluorescent; however, in the presence of NO and oxygen, it forms DAF-2 triazole (DAF-2 T), a fluorescent product. This approach allows the direct visualization and semi-quantitative analysis of the basal NO availability at the cell level. Excess dye was removed by washing out the dye with a bath solution. The cells were imaged in Hanks buffer (pH 7.4). [NO]c was assessed by a confocal scanning laser microscope (Leica TCS SP5). DAF-2T fluorescence was excited with the 488 nm line of an argon ion laser and the emitted fluorescence was measured at 515 nm. Time course software was used to capture images of the cells at 2-second intervals (xyt) in the Live Data Mode acquisition. By applying the LSCM computer software, the intensities of the intracellular maximum or minimum fluorescence were measured. From these data, the initial fluorescence value at t = 0 s was taken as F0, and the final fluorescence intensity value F was registered after TERPY (10 lM) addition (at 2 s). In this way, positive values from D%FI reflect the increase in [NO]c, which was obtained for each protocol in relation to F (100%). To assess basal [NO]c, values from F0 were measured.
D%FI ¼ ðF F 0 =F Þ 100 HPLC analysis Dihydroethidium (DHE) is a widely used sensitive superoxide (O 2 ) probe. 2-Hydroxyethidium (EOH) and ethidium (E) are products of DHE oxidation. However, EOH is more specific for O 2 than the less-specific product E. Separation of DHE, EOH and E was performed as described previously [32]. The chromatographic separation was carried out using a NovaPak C18 column (3.9 150 mm, 5 lm particle size) in an HPLC system (Waters) equipped with a rheodyne injector and photodiode array (W2996) and fluorescence (W2475) detectors, using solution A (pure acetonitrile) and solution B (water/10% acetonitrile/0.1% trifluoracetic acid) as the mobile phase at a flow rate of 0.4 mL min1. Runs were started with 0% A, increased linearly to 40% A during the initial 10 min, kept at this 7 proportion for an additional 10 min, changed to 100% A for an additional 5 min, and back to 0% A for the final 10 min. DHE was monitored by UV absorption at 245 nm. EOH was monitored by fluorescence detection with excitation at 510 nm and emission at 595 nm. Quantification was performed by comparison of integrated peak areas of the obtained and standard solutions under identical chromatographic conditions.
HPLC analysis of superoxide in aortic segments Aortic extraction was performed as described previously [32], with the specified modifications. Aortic artery segments (3 mm in length) were incubated in 0.5 mL of PBS/DTPA, in the presence or absence of vitamin C (100 lM), for 20 min, in a 1.5 mL Eppendorf vial. A volume 2.5 lL of DHE 10 mM stock solution was added to the buffer, to achieve a final concentration of 50 lM, and a final DMSO concentration of 0.5% v/v. Incubation was carried out for 30 min at 37 °C in the dark. Artery segments were washed in PBS, transferred to liquid nitrogen, and homogenized with mortar and pestle. The homogenate was resuspended in acetonitrile (0.5 mL), sonicated (3 cycles at 8 W for 10 s), and centrifuged (12,000g for 10 min at 4 °C). Supernatants were dried under vacuum (Speed VacR Plus SC-110A, Thermo Savant) and pellets were maintained at 20 °C, in the dark, until analysis was carried out. Samples were suspended in 120 lL PBS/DTPA and injected (100 lL) into the HPLC system. The simultaneous detection of DHE and its derived oxidation product (EOH) using ultraviolet and fluorescence detection, respectively, allowed the ideal situation for using DHE as an internal control during the organic extraction of each sample. Thus, DHEderived products were expressed as the ratio of EOH generated per DHE consumed (initial 50 lM DHE concentration minus remaining DHE; nmol EOH/lmol DHE).
Materials The composition of PSS was the following (in mM): 130.0 NaCl, 1.6 CaCl2, 4.7 KCl, 1.17 MgSO4, 1.18 KH2PO4, 14.9 NaHCO3, 0.026 EDTA, and 5.5 dextrose. Acetylcholine, phenylephrine, Fluo 3-AM, collagenase Type II-S, bovine serum albumin (type I), dimethyl sulfoxide (Grade I) DMSO, poly-L-lysine solution, DAF-2 DA, and vitamin C were obtained from Sigma–Aldrich (St. Louis, MO, USA). DHE was purchased from Invitrogen (Carlsbad, CA). The [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) complex was synthesized as previously described [21]. It was synthesized at the Laboratory of Analytical Chemistry by Dr. Roberto Santana da Silva.
Statistical analysis Data are expressed as means ± S.E.M. In each set of experiments, n indicates the number of rats studied. The values for vascular reactivity and responses to TERPY are expressed as percentage of the preceding contraction induced by phenylephrine. The concentration of the agonist producing a half-maximal response (EC50) was determined after logit transformation of the normalized concentration–response curves and it is reported as the negative logarithm (log EC50 = pD2 values) of the mean of individual values for each tissue, using GraphPad Prism version 3.0 (GraphPad Software Inc., San Diego, CA). The maximum relaxant effect (ME) was considered as the maximal amplitude response reached in concentration–effect curves for relaxant agents. The decrease in [Ca2+]c in cells stimulated with TERPY was obtained from D%FI. The statistical analysis of the results was performed by using GraphPad Prism version 3.0. The statistical significance was tested by one-way ANOVA (post-test: Newman–Keuls) and Student’s t test, and values of P < 0.05 were considered significant.
Results Functional studies The new NO donor (TERPY 1 nM to 300 lM) induces a concentration-dependent relaxation in endotheliumdenuded aortas contracted with phenylephrine (Fig. 1). The potency of TERPY in inducing relaxation is greater in the aortic ring from 2K rats (pD2: 7.08 ± 0.06, n = 7) than in 2K-1C (pD2: 6.55 ± 0.07, p < 0.001, n = 7), whereas
Fig. 1. Effects of vitamin C (Vit. C––100 lM)) on TERPY (10 lM) induced relaxation in the aortic rings from normotensive (2K) and hypertensive (2K-1C) rats. Concentration–response curves to [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) in denuded aortic rings precontracted with phenylephrine. Values are means ± S.E.M. and are expressed as percentage of relaxation to TERPY.
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the maximum relaxant effect is similar when 2K (109.3 ± 1.8%) and 2K-1C (110.5 ± 2.1%) are compared (Fig. 1). Addition of the antioxidant vitamin C (100 lM) improves the relaxation induced by TERPY in the aortic rings from 2K-1C. Vitamin C increases the potency of TERPY in 2K-1C aortas (pD2: 6.97 ± 0.04, n = 6), but it does not modify the maximum relaxant effect to TERPY in the aortic rings from 2K-1C (115.5 ± 4.9%). On the other hand, vitamin C does not modify the potency (pD2: 6.97 ± 0.10, n = 6) or the maximum relaxant effect (105.6 ± 2.2%) to TERPY in the aortic rings from 2K rats (Fig. 1).
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from 2K (%DFI: 58.47 ± 4.83%, n = 5) than in those from 2K-1C (%DFI: 35.54 ± 2.38%, p < 0.001, n = 4), as shown in Fig. 2B. These results support the vascular reactivity study data using TERPY (1 nM to 300 lM) in 2K and 2K-1C, as shown in Fig. 1. Addition of the antioxidant vitamin C (100 lM) increases the effect of TERPY (10 lM) with regard to the [Ca2+]c decrease in cells from 2K-1C (D%FI: 47.58 ± 3.77%, n = 4) rats, as shown in Fig. 2B. However, vitamin C does not change the effect of TERPY on [Ca2+]c in 2K aortic cells (D%FI: 58.69 ± 5.78%, n = 5) (Fig. 2B). These results support the vascular reactivity study data using TERPY in 2K and 2K-1C aortic rings treated with vitamin C (Fig. 1).
Ca2+ measurements NO measurements Using the confocal microscope, we examined the effect of TERPY on [Ca2+]c in VSMCs from 2K and 2K-1C rat aortas, pre-loaded with Fluo 3-AM. As shown in Fig. 2A, a decrease in the fluorescence of Fluo 3 in VSMCs from 2K rats can be visualized in the x–y scanned images after the addition of TERPY (at 115 s). The decrease in [Ca2+]c in response to TERPY (10 lM) is sharper in cells
We used the fluorescent NO indicator DAF-2 DA to measure [NO]c in the VSMCs from the rat aorta. The NO basal levels inside the cells from 2K-1C (F0: 34.46 ± 1.73, n = 3) were lower than the levels obtained in the in 2K aortic cells (F0: 105.37 ± 1.54, p < 0.001, n = 3) (Fig. 3A).
Fig. 2. Decrease of cytosolic Ca2+ concentration ([Ca2+]c) induced by [Ru(NH.NHq)(terpy)NO+]3+ (TERPY––10 lM) in normotensive (2K) and renal hypertensive (2K-1C) vascular smooth muscle cells (VSMCs) from rat aortas. Cells were preloaded with Fluo-3 AM (10 lmol/L) and stimulated with TERPY (10 lmol/L). (A) Temporal effect of TERPY in a single vascular smooth muscle cell was recorded in the confocal microscope. TERPY was added after 115 s. The intensities of the colors indicate higher [Ca2+]c (as shown in the left bar). (B) Effect of vitamin C (Vit. C––100 lM)) on the decrease in [Ca2+]c induced by TERPY in VSMCs from 2K and 2K-1C rats. D%FI indicates the average of % [Ca2+]c reduction. Differences between D%FI values are indicated as ***p < 0.001, 2K-1C versus 2K and *p < 0.05, 2K-1C versus 2K-1C + Vit. C.
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Fig. 3. Cytosolic NO concentration ([NO]c) in vascular smooth muscle cells (VSMCs) from normotensive (2K) and renal hypertensive (2K-1C) rat aortas, preloaded with DAF-2 DA (5 lM). (A) The basal [NO]c values were obtained from F0 (initial fluorescence intensity). (B) Serial NO images of DAF-2T fluorescence in a single vascular smooth muscle cell were recorded after the addition of TERPY (10 lM) at 2 s. The intensity of the colors indicate higher [NO]c (as shown in the left bar). (C) Effect of TERPY on [NO]c in cells indicated by D%FI. Differences in F0 and D%FI values to FI are indicated as *** p < 0.001, 2K-1C versus 2K.
As shown in Fig. 3B, an increase in the fluorescence of DAF-2T in VSMCs from 2K rats can be visualized in the x–y scanned images after addition of TERPY (10 lM at 2 s). In Fig. 3C, the increase in [NO]c in response to TERPY (10 lM) is more pronounced in cells from 2K (D%FI: 19.76 ± 1.65%, n = 4) than in those from 2K-1C (D%FI: 10.17 ± 0.61%, p < 0.001, n = 4). Superoxide measurements in aortic segments The concentration of O 2 was measured by analyzing the DHE-derived oxidation products in the aortic segments. HPLC analysis revealed increased basal levels of the EOH/DHE ratio in the aortic tissue from 2K-
1C rats (33.39 ± 8.37 nmol/lmol, n = 5), which is higher than the ratio found in the aortic tissue from 2K rats (7.76 ± 1.59 nmol/lmol, n = 7) (Fig. 4). This shows that the aortic segments from 2K-1C rats have a higher concentration of O compared with the 2K aortic 2 segments. In the rat aortic segments incubated with vitamin C (100 lM), HPLC analysis revealed that the EOH/DHE ratio is lower in aortic segments from 2K-1C rats (7.53 ± 2.99 nmol/lmol, n = 5) but it is not modified in the aortic segments from 2K rats (9.21 ± 1.59 nmol/lmol, n = 6) (Fig. 4). We observed that vitamin C (100 lM) normalizes the O 2 concentration in the aortic segments from 2K-1C rats.
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Fig. 4. Concentration of the superoxide radical (O 2 ) in the aortic segments as indicated by 2-hydroxyethidium (EOH)/dihydroethidium (DHE) ratio. These segments (3 mm in length) were incubated in the presence or in the absence of vitamin C (Vit. C––100 lM). The DHEderived product was expressed as ratio of EOH generated per DHE consumed (initial 50 lM DHE concentration minus remaining DHE; nmol EOH/lmol DHE). Differences in the EOH/DHE ratio values are indicated as ***p < 0.001, 2K-1C versus 2K, 2K + Vit. C, 2K-1C + Vit. C.
Discussion The main finding of the present study is that the impaired effect of the new NO donor (TERPY) on the aortas from 2K-1C rats is normalized by the antioxidant agent vitamin C. Therefore, our results provide clear evidence of the involvement of the superoxide in the impaired effect of TERPY in the aortas from hypertensive 2K-1C rats. We have found decreased sensitivity to the NO donor TERPY in the aortic rings from 2K-1C rats compared to normotensive 2K rats. In addition, the reduction in [Ca2+]c in response to TERPY is impaired in VSMCs from 2K-1C rats. In previous studies carried out in our laboratory, we also reported a reduction in the sensitivity to NO in the aortas from 2K-1C rats, although this was done for different NO donors [5,6]. Some authors have attributed this impairment to an increased O 2 production in the aorta from hypertensive rats [10,20,32], since NO reacts with O 2 and forms peroxynitrite (ONOO ), thereby reduc ing NO bioavailability. The major O2 source in VSMCs is the NAD(P)H oxidase, which is stimulated by angiotensin II [33]. It is well recognized that the development and maintenance of Goldblatt hypertension 2K-1C is dependent on the renin–angiotensin system [34]. Rajagoplan et al. (1996) have demonstrated that angiotensin II infusion can cause hypertension and increase in the vascular O 2 production secondary to the activation of NAD(P)H-dependent oxidases. In this way, elevated angiotensin II levels could be activating NAD(P)H oxidase and increasing O 2 in the VSMCs from 2K-1C rats. We used vitamin C as a pharmacological tool to investigate whether the impaired effect to TERPY in the aorta from 2K-1C rats could be due to elevated O 2 concentrations. Our results show that the relaxant effect to TERPY is normalized in the aortic rings from 2K-1C rats incubated with vitamin C (100 lM). In the same way, the decrease in
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[Ca2+]c in response to TERPY was normalized in VSMCs from 2K-1C rats incubated with vitamin C (100 lM). This treatment does not modify the effect to TERPY in the aortas and VSMCs from 2K rats. Therefore, it can be suggested that vitamin C does not modify the NO release from TERPY, but it reduces the O 2 levels in the aortic segments from 2K-1C rats. The free radical-scavenging effect of antioxidant vitamins such as vitamin C is well documented [35]. Also, some studies have shown that the antioxidant properties of vitamin C are associated with the decreased activation of NAD(P)H oxidase and the increased activity of superoxide dismutase, which is an antioxidant enzyme that metabolizes free radicals [20]. In addition, as reported by Newaz et al. (2005) there is an increase in nitric oxide synthase (NOS) activity with addition of dietary vitamin C [36]. In the present study, our results indicate that vitamin C increases the effect of NO on aorta from hypertensive rats. According to Bonaventura et al. (2007), the relaxation induced by TERPY is due to release of radicalar NO (NO ) and nitroxyl anion (NO). Therefore, we should consider the possibility that vitamin C also scavenges the NO. However, our results suggest that it is not the case because the incubation of the aortic rings with vitamin C did not change the effects of TERPY in the isolated aortas and VSMCs from 2K rat aortas. The NO basal levels were measured using a fluorescent NO probe (DAF-2 DA) in the confocal microscope. We verified lower intracellular NO basal levels in the aortic VSMCs from 2K-1C rats. In the same way, the increase in [NO]c in response to TERPY is lower in the aortic VSMCs from 2K-1C rats when compared with 2K rats. Therefore, we could verify that the NO bioavailability is reduced in VSMCs from 2K-1C rats, which could account for the impaired effect to TERPY in the aorta from 2K-1C. It was not possible to measure the [NO] in presence of vitamin C, since it is a quenching agent for DAF-2T reaction product of NO and O2 [31]. Superoxide quantification in the aortic segments was performed by HPLC analysis. The O 2 concentration is higher in the aortas from 2K-1C rats than those from 2K rats. Nevertheless, vitamin C normalizes the O 2 concentration in the aortas from 2K-1C rats. These results agree with those of other studies, which have described increased O 2 concentrations in the aortas from renovascular hypertensive rats [8–10]. In the same way, other authors have shown that the treatment with antioxidants normalizes O 2 concentration in the aortas from hypertensive rats [8,10,20,26]. In conclusion, our results show that the impaired effect to TERPY in the aortas from 2K-1C rats is due to increased O 2 concentration that is normalized by vitamin C. Acknowledgments We thank Ma´rcia C.Z. Graeff for technical assistance in the confocal microscopy (Faculdade de Medicina de Ri-
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Nitric Oxide 18 (2008) 176–183 www.elsevier.com/locate/yniox
Vitamin C improves the effect of a new nitric oxide donor on the vascular smooth muscle from renal hypertensive rats G.J. Rodrigues a, C.N. Lunardi a, R.G. Lima a, C.X. Santos b, F.R.M. Laurindo b, R.S. da Silva a, L.M. Bendhack a,* a
Departamento de Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, USP, 14040-903 Ribeira˜o Preto, SP, Brazil b Laborato´rio de Biologia Vascular, Instituto do Coracßa˜o (InCor), USP, 05403-000, Brazil Received 14 September 2007; revised 29 November 2007 Available online 25 December 2007
Abstract Impaired relaxation induced by the new nitric oxide (NO) donor [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) has been observed in the aortic rings from renal hypertensive rats (2K-1C). An increased production of reactive oxygen species (ROS) in the aortas from 2K1C rats are capable of reducing NO bioavailability. Therefore, this study aimed at investigating the effects of an antioxidant (vitamin C) on the relaxant effect of NO released from TERPY on the 2K-1C rat aorta. As for vascular reactivity, the potency of TERPY is greater in the control rats (2K) than in 2K-1C whereas the maximum relaxation (ME) is not significantly different between the 2K and 2K-1C rat aortas. The relaxation of TERPY is potentiated only in the 2K-1C aortic ring treated with vitamin C. TERPY has a lower effect in decreasing cytosolic Ca2+ concentration ([Ca2+]c) in vascular smooth muscle cells (VSMCs) from 2K-1C rats. This effect is also potentiated in 2K-1C aortic cells treated with vitamin C, but it is not altered in 2K cells. The basal cytosolic NO concentration ([NO]c) is lower in 2K-1C than in 2K cells, and the bioavailability of the NO released from TERPY is larger in 2K than in 2K-1C VSMCs. The superoxide radical concentration ([O 2 ]) is higher in the 2K-1C aorta, and vitamin C reduces the [O2 ] in the 2K-1C aorta. Taken together, these results show that in the aortas of renal hypertensive 2K-1C rats, released NO from the new NO donor is not available to produce a similar effect in 2K aorta due to increased [O 2 ]. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Nitric oxide donor; Superoxide anion; Renal hypertension; Antioxidant
Introduction Nitric oxide (NO) regulates an array of physiological processes including the maintenance of vascular tone. The vasorelaxation induced by NO involves mainly the activation of soluble guanylyl cyclase, which catalyzes the conversion of guanosine triphosphate (GTP) into cyclic guanosine monophosphate [1]. Acting as a second messenger, cGMP activates a family of serine/threonine protein kinases called cGMP-dependent protein kinases (PKGs), which induce relaxation by decrease of cytosolic Ca2+ concentration ([Ca2+]c) and Ca2+ desensitization of actin–
*
Corresponding author. Fax: +55 16 36024880. E-mail address: [email protected] (L.M. Bendhack).
1089-8603/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2007.12.002
myosin contractile system, in vascular smooth muscle cells [2]. Various cGMP-independent mechanisms of NO have been reported in a number of studies, including the direct activation of K+ channels [3,4], as well the activation of Na+/K+-ATPase pump [4]. In previous studies, we have observed an impaired relaxation to NO in arteries from renovascular hypertensive rats [5,6]. This impairment has been attributed to the increased production of reactive oxygen species (ROS) [7]. The superoxide (O 2 ) is an important member of the ROS family, which has been found in elevated concentration in the vascular smooth muscle cells from renal hypertensive rats [8– 10]. The NO bioavailability could be significantly reduced in the presence of O 2 because NO reacts with O2 to form peroxynitrite (ONOO ) [11]. These potentially harmful vascular effects of the superoxide are normally counterbal-
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anced by the enzyme superoxide dismutase and other antioxidants such as vitamins E and C [12–16]. An imbalance between the production of ROS and the level of protective antioxidants could lead to significant increases in the oxidative stress and arterial pressure [11]. A restoration of this balance by antioxidant oral treatment with tempol [17,18], sesamin [17,19], vitamin E [19,20], and vitamin C [20] has been shown to decrease the arterial pressure in hypertensive rats. NO donors are pharmacologically active substances that release NO in vivo or in vitro. [Ru(terpy)(bdq)NO+]3+ (TERPY) is a nitrosyl ruthenium complex that is thermally stable in physiological pH and it has been described as an NO donor [21] that induces vascular relaxation. The cellular mechanisms involved in this effect have been described by Bonaventura et al. [22]. This compound is attractive since NO donors used in clinical medicine present some disadvantages. For instance, the chronic treatment with nitroglycerin induces tolerance [23], and sodium nitroprusside releases cyanide [24], which is toxic to the vascular cells. Therefore, the aim of this study was to verify the influence of the antioxidant agent vitamin C on the effect of the new NO donor TERPY on the aortic vascular smooth muscle from renal hypertensive rats (2K-1C). Materials and methods Experimental animals Experimental protocols followed standards and policies of the Animal Care and Use Committee of the University of Sa˜o Paulo. Renovascular hypertension was induced in rats following the 2K-1C Goldblatt model [25]. Briefly, male Wistar rats (180–200 g) were anesthetized with tribromoethanol (2.5 mg kg1, i.p.) and after a midline laparotomy a silver clip with an internal diameter of 0.20 mm was placed around the left renal artery. Normotensive two-kidney rats (2K) were only submitted to laparotomy. Animals were maintained on standard rat chow with a 12 h light/dark cycle and given free access to both food (standard rat chow) and water. The systolic blood pressure (SBP) was measured weekly in non-anesthetized animals by an indirect tail-cuff method (MLT125R pulse transducer/pressure cuff coupled to the PowerLab 4/S analog-to-digital converter; AD Instruments Pty Ltd., Castle Hill, Australia), and rats were considered hypertensive when the SBP was higher than 160 mmHg.
Functional studies Rats were killed by decapitation six weeks after surgery, and the thoracic aortas were isolated. Aortic rings, 4 mm in length, were placed in bath chambers (10 mL) for isolated organs containing physiological salt solution (PSS) at 37 °C, continuously bubbled with 95% O2 and 5% CO2, pH 7.4. Two fine stainless steel holders were placed through the lumen of the aortic rings, one of the holders was fixed to the tissue chamber and the other was connected to an F-60 force–displacement transducer. The contractile/relaxant responses were recorded on a polygraph (Narco Biosystems Inc., Houston, TX, USA). The aortic rings were submitted to a tension of 1.5 g, which was readjusted every 15 min during a 60 min equilibration period before addition of the given drug. An optimal basal tension of 1.5 g in the aortic rings from both 2K and 2K-1C was previously standardized by exposing the vessels to 90 mM KCl under various resting tensions (0.25–2.5 g). The endothelium was mechanically removed by gently rubbing the intimal surface with stainless steel holders. Endothelial integrity was qualitatively assessed by the degree of relaxation caused
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by 1 lM acetylcholine in the presence of contractile tone induced by phenylephrine (0.1 lM). As our studies required endothelium denuded aortas, the rings were discarded if there was any degree of relaxation, to avoid the possible influence of endothelial factors. After endothelial integrity was assessed, aortic rings were pre-contracted with phenylephrine (0.1 lM), and concentration–effect curves to NO donor TERPY (1 nM to 300 lM) were constructed in the 2K and 2K-1C aortic rings when the plateau was reached. Vitamin C (100 lM) [26] was added 20 min before phenylephrine. Concentration–effect curves to TERPY (1 nM to 300 lM) were constructed in order to examine the effect of vitamin C on the relaxation induced by the NO donor, in the aortic rings isolated from 2K and 2K-1C rats pre-contracted with phenylephrine (0.1 lM), in the presence or absence of vitamin C.
Vascular smooth muscle cells isolation Rats were killed by decapitation six weeks after surgery, and the thoracic aortas were isolated. Vascular smooth muscle cells (VSMCs) were isolated by enzymatic digestion from aorta as described previously [29]. Briefly, the aortas were dissected and longitudinally opened. The endothelium and the adventitia were removed, and the tissue was minced into small pieces and incubated in Ca2+ and Mg+ free Hanks solution with the following composition (in mM): 145.0 NaCl, 5.0 KCl, 0.5 NaH2PO4, 10.0 dextrose and 10.0 HEPES (pH 7.4) containing 0.03 mg/mL collagenase Type II-S. The tissue was gently shaken in this solution for 25 min at 37 °C and bubbled with carbogen mixture. After that, 10 mg/mL bovine serum albumin (type I) was added to the vessel fragments in Ca2+ and Mg+ free Hanks solution, and cells were released by mechanical dispersion with a Pasteur pipette. The resultant cell suspension was centrifuged at 1000 rpm for 3 min and suspended in Ca2+ and Mg+ free Hanks. The cells were plated on glass coverslips pretreated with poly-L-lysine solution and kept in a humidified 37 °C incubator gassed with 5% CO2. The cells were used 3 h after plating and were maintained in a serum-free medium. A similar procedure was carried out for 2K and 2K-1C rats.
Ca2+ measurements To confirm the results obtained with TERPY in the aorta tissue by vascular reactivity studies, we used confocal microscopy. In order to assess the transient of the cytosolic Ca2+ concentration ([Ca2+]c), VSMCs were loaded with Fluo 3-AM (10 lM) for 30 min at room temperature. Excess dye was removed by washing out the dye with bath solution and allowing 30 min for intracellular desterification of Fluo 3-AM to Fluo 3. The cells were imaged in Hanks (with the following composition in mM: 1.6 CaCl2, 1.0 MgCl2, 145.0 NaCl, 5.0 KCl, 0.5 NaH2PO4, 10.0 dextrose and 10.0 HEPES, pH 7.4) and [Ca2+]c was assessed by a confocal scanning laser microscope (Leica TCS SP5) [27–30]. Fluo 3 fluorescence was excited with the 488 nm line of an argon ion laser and the emitted fluorescence was measured at 510 nm. A time course software was used to capture images of the cells at intervals of 0.850 s (xyt) in Live Data Mode acquisition. By applying the computer software of the LSCM, the intensities of the intracellular maximum or minimum fluorescence were measured. From this data, it was used as the initial fluorescence intensity value at t = 0 s was taken as F0, and the final value of the fluorescence intensity F was registered after TERPY (10 lM) addition. In this way, negative values from differences in percentage of fluorescence intensity (D%FI) reflects the decrease in [Ca2+]c, which was obtained for each protocol in relation to basal (F0) (100%). Vitamin C (100 lM) was added 20 min before TERPY (10 lM).
D%FI ¼ ðF F 0 =F 0 Þ 100 NO measurements To assess the cytosolic nitric oxide concentration ([NO]c), VSMCs were loaded with the NO indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) (5 lM) for 30 min, at room temperature [31]. The membrane
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permeable DAF-2 DA readily enters the cells and is subsequently hydrolyzed by cytosolic esterases releasing free DAF-2, which does not leak into the medium. At physiological pH, DAF-2 is relatively non-fluorescent; however, in the presence of NO and oxygen, it forms DAF-2 triazole (DAF-2 T), a fluorescent product. This approach allows the direct visualization and semi-quantitative analysis of the basal NO availability at the cell level. Excess dye was removed by washing out the dye with a bath solution. The cells were imaged in Hanks buffer (pH 7.4). [NO]c was assessed by a confocal scanning laser microscope (Leica TCS SP5). DAF-2T fluorescence was excited with the 488 nm line of an argon ion laser and the emitted fluorescence was measured at 515 nm. Time course software was used to capture images of the cells at 2-second intervals (xyt) in the Live Data Mode acquisition. By applying the LSCM computer software, the intensities of the intracellular maximum or minimum fluorescence were measured. From these data, the initial fluorescence value at t = 0 s was taken as F0, and the final fluorescence intensity value F was registered after TERPY (10 lM) addition (at 2 s). In this way, positive values from D%FI reflect the increase in [NO]c, which was obtained for each protocol in relation to F (100%). To assess basal [NO]c, values from F0 were measured.
D%FI ¼ ðF F 0 =F Þ 100 HPLC analysis Dihydroethidium (DHE) is a widely used sensitive superoxide (O 2 ) probe. 2-Hydroxyethidium (EOH) and ethidium (E) are products of DHE oxidation. However, EOH is more specific for O 2 than the less-specific product E. Separation of DHE, EOH and E was performed as described previously [32]. The chromatographic separation was carried out using a NovaPak C18 column (3.9 150 mm, 5 lm particle size) in an HPLC system (Waters) equipped with a rheodyne injector and photodiode array (W2996) and fluorescence (W2475) detectors, using solution A (pure acetonitrile) and solution B (water/10% acetonitrile/0.1% trifluoracetic acid) as the mobile phase at a flow rate of 0.4 mL min1. Runs were started with 0% A, increased linearly to 40% A during the initial 10 min, kept at this 7 proportion for an additional 10 min, changed to 100% A for an additional 5 min, and back to 0% A for the final 10 min. DHE was monitored by UV absorption at 245 nm. EOH was monitored by fluorescence detection with excitation at 510 nm and emission at 595 nm. Quantification was performed by comparison of integrated peak areas of the obtained and standard solutions under identical chromatographic conditions.
HPLC analysis of superoxide in aortic segments Aortic extraction was performed as described previously [32], with the specified modifications. Aortic artery segments (3 mm in length) were incubated in 0.5 mL of PBS/DTPA, in the presence or absence of vitamin C (100 lM), for 20 min, in a 1.5 mL Eppendorf vial. A volume 2.5 lL of DHE 10 mM stock solution was added to the buffer, to achieve a final concentration of 50 lM, and a final DMSO concentration of 0.5% v/v. Incubation was carried out for 30 min at 37 °C in the dark. Artery segments were washed in PBS, transferred to liquid nitrogen, and homogenized with mortar and pestle. The homogenate was resuspended in acetonitrile (0.5 mL), sonicated (3 cycles at 8 W for 10 s), and centrifuged (12,000g for 10 min at 4 °C). Supernatants were dried under vacuum (Speed VacR Plus SC-110A, Thermo Savant) and pellets were maintained at 20 °C, in the dark, until analysis was carried out. Samples were suspended in 120 lL PBS/DTPA and injected (100 lL) into the HPLC system. The simultaneous detection of DHE and its derived oxidation product (EOH) using ultraviolet and fluorescence detection, respectively, allowed the ideal situation for using DHE as an internal control during the organic extraction of each sample. Thus, DHEderived products were expressed as the ratio of EOH generated per DHE consumed (initial 50 lM DHE concentration minus remaining DHE; nmol EOH/lmol DHE).
Materials The composition of PSS was the following (in mM): 130.0 NaCl, 1.6 CaCl2, 4.7 KCl, 1.17 MgSO4, 1.18 KH2PO4, 14.9 NaHCO3, 0.026 EDTA, and 5.5 dextrose. Acetylcholine, phenylephrine, Fluo 3-AM, collagenase Type II-S, bovine serum albumin (type I), dimethyl sulfoxide (Grade I) DMSO, poly-L-lysine solution, DAF-2 DA, and vitamin C were obtained from Sigma–Aldrich (St. Louis, MO, USA). DHE was purchased from Invitrogen (Carlsbad, CA). The [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) complex was synthesized as previously described [21]. It was synthesized at the Laboratory of Analytical Chemistry by Dr. Roberto Santana da Silva.
Statistical analysis Data are expressed as means ± S.E.M. In each set of experiments, n indicates the number of rats studied. The values for vascular reactivity and responses to TERPY are expressed as percentage of the preceding contraction induced by phenylephrine. The concentration of the agonist producing a half-maximal response (EC50) was determined after logit transformation of the normalized concentration–response curves and it is reported as the negative logarithm (log EC50 = pD2 values) of the mean of individual values for each tissue, using GraphPad Prism version 3.0 (GraphPad Software Inc., San Diego, CA). The maximum relaxant effect (ME) was considered as the maximal amplitude response reached in concentration–effect curves for relaxant agents. The decrease in [Ca2+]c in cells stimulated with TERPY was obtained from D%FI. The statistical analysis of the results was performed by using GraphPad Prism version 3.0. The statistical significance was tested by one-way ANOVA (post-test: Newman–Keuls) and Student’s t test, and values of P < 0.05 were considered significant.
Results Functional studies The new NO donor (TERPY 1 nM to 300 lM) induces a concentration-dependent relaxation in endotheliumdenuded aortas contracted with phenylephrine (Fig. 1). The potency of TERPY in inducing relaxation is greater in the aortic ring from 2K rats (pD2: 7.08 ± 0.06, n = 7) than in 2K-1C (pD2: 6.55 ± 0.07, p < 0.001, n = 7), whereas
Fig. 1. Effects of vitamin C (Vit. C––100 lM)) on TERPY (10 lM) induced relaxation in the aortic rings from normotensive (2K) and hypertensive (2K-1C) rats. Concentration–response curves to [Ru(NH.NHq)(terpy)NO+]3+ (TERPY) in denuded aortic rings precontracted with phenylephrine. Values are means ± S.E.M. and are expressed as percentage of relaxation to TERPY.
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the maximum relaxant effect is similar when 2K (109.3 ± 1.8%) and 2K-1C (110.5 ± 2.1%) are compared (Fig. 1). Addition of the antioxidant vitamin C (100 lM) improves the relaxation induced by TERPY in the aortic rings from 2K-1C. Vitamin C increases the potency of TERPY in 2K-1C aortas (pD2: 6.97 ± 0.04, n = 6), but it does not modify the maximum relaxant effect to TERPY in the aortic rings from 2K-1C (115.5 ± 4.9%). On the other hand, vitamin C does not modify the potency (pD2: 6.97 ± 0.10, n = 6) or the maximum relaxant effect (105.6 ± 2.2%) to TERPY in the aortic rings from 2K rats (Fig. 1).
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from 2K (%DFI: 58.47 ± 4.83%, n = 5) than in those from 2K-1C (%DFI: 35.54 ± 2.38%, p < 0.001, n = 4), as shown in Fig. 2B. These results support the vascular reactivity study data using TERPY (1 nM to 300 lM) in 2K and 2K-1C, as shown in Fig. 1. Addition of the antioxidant vitamin C (100 lM) increases the effect of TERPY (10 lM) with regard to the [Ca2+]c decrease in cells from 2K-1C (D%FI: 47.58 ± 3.77%, n = 4) rats, as shown in Fig. 2B. However, vitamin C does not change the effect of TERPY on [Ca2+]c in 2K aortic cells (D%FI: 58.69 ± 5.78%, n = 5) (Fig. 2B). These results support the vascular reactivity study data using TERPY in 2K and 2K-1C aortic rings treated with vitamin C (Fig. 1).
Ca2+ measurements NO measurements Using the confocal microscope, we examined the effect of TERPY on [Ca2+]c in VSMCs from 2K and 2K-1C rat aortas, pre-loaded with Fluo 3-AM. As shown in Fig. 2A, a decrease in the fluorescence of Fluo 3 in VSMCs from 2K rats can be visualized in the x–y scanned images after the addition of TERPY (at 115 s). The decrease in [Ca2+]c in response to TERPY (10 lM) is sharper in cells
We used the fluorescent NO indicator DAF-2 DA to measure [NO]c in the VSMCs from the rat aorta. The NO basal levels inside the cells from 2K-1C (F0: 34.46 ± 1.73, n = 3) were lower than the levels obtained in the in 2K aortic cells (F0: 105.37 ± 1.54, p < 0.001, n = 3) (Fig. 3A).
Fig. 2. Decrease of cytosolic Ca2+ concentration ([Ca2+]c) induced by [Ru(NH.NHq)(terpy)NO+]3+ (TERPY––10 lM) in normotensive (2K) and renal hypertensive (2K-1C) vascular smooth muscle cells (VSMCs) from rat aortas. Cells were preloaded with Fluo-3 AM (10 lmol/L) and stimulated with TERPY (10 lmol/L). (A) Temporal effect of TERPY in a single vascular smooth muscle cell was recorded in the confocal microscope. TERPY was added after 115 s. The intensities of the colors indicate higher [Ca2+]c (as shown in the left bar). (B) Effect of vitamin C (Vit. C––100 lM)) on the decrease in [Ca2+]c induced by TERPY in VSMCs from 2K and 2K-1C rats. D%FI indicates the average of % [Ca2+]c reduction. Differences between D%FI values are indicated as ***p < 0.001, 2K-1C versus 2K and *p < 0.05, 2K-1C versus 2K-1C + Vit. C.
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Fig. 3. Cytosolic NO concentration ([NO]c) in vascular smooth muscle cells (VSMCs) from normotensive (2K) and renal hypertensive (2K-1C) rat aortas, preloaded with DAF-2 DA (5 lM). (A) The basal [NO]c values were obtained from F0 (initial fluorescence intensity). (B) Serial NO images of DAF-2T fluorescence in a single vascular smooth muscle cell were recorded after the addition of TERPY (10 lM) at 2 s. The intensity of the colors indicate higher [NO]c (as shown in the left bar). (C) Effect of TERPY on [NO]c in cells indicated by D%FI. Differences in F0 and D%FI values to FI are indicated as *** p < 0.001, 2K-1C versus 2K.
As shown in Fig. 3B, an increase in the fluorescence of DAF-2T in VSMCs from 2K rats can be visualized in the x–y scanned images after addition of TERPY (10 lM at 2 s). In Fig. 3C, the increase in [NO]c in response to TERPY (10 lM) is more pronounced in cells from 2K (D%FI: 19.76 ± 1.65%, n = 4) than in those from 2K-1C (D%FI: 10.17 ± 0.61%, p < 0.001, n = 4). Superoxide measurements in aortic segments The concentration of O 2 was measured by analyzing the DHE-derived oxidation products in the aortic segments. HPLC analysis revealed increased basal levels of the EOH/DHE ratio in the aortic tissue from 2K-
1C rats (33.39 ± 8.37 nmol/lmol, n = 5), which is higher than the ratio found in the aortic tissue from 2K rats (7.76 ± 1.59 nmol/lmol, n = 7) (Fig. 4). This shows that the aortic segments from 2K-1C rats have a higher concentration of O compared with the 2K aortic 2 segments. In the rat aortic segments incubated with vitamin C (100 lM), HPLC analysis revealed that the EOH/DHE ratio is lower in aortic segments from 2K-1C rats (7.53 ± 2.99 nmol/lmol, n = 5) but it is not modified in the aortic segments from 2K rats (9.21 ± 1.59 nmol/lmol, n = 6) (Fig. 4). We observed that vitamin C (100 lM) normalizes the O 2 concentration in the aortic segments from 2K-1C rats.
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Fig. 4. Concentration of the superoxide radical (O 2 ) in the aortic segments as indicated by 2-hydroxyethidium (EOH)/dihydroethidium (DHE) ratio. These segments (3 mm in length) were incubated in the presence or in the absence of vitamin C (Vit. C––100 lM). The DHEderived product was expressed as ratio of EOH generated per DHE consumed (initial 50 lM DHE concentration minus remaining DHE; nmol EOH/lmol DHE). Differences in the EOH/DHE ratio values are indicated as ***p < 0.001, 2K-1C versus 2K, 2K + Vit. C, 2K-1C + Vit. C.
Discussion The main finding of the present study is that the impaired effect of the new NO donor (TERPY) on the aortas from 2K-1C rats is normalized by the antioxidant agent vitamin C. Therefore, our results provide clear evidence of the involvement of the superoxide in the impaired effect of TERPY in the aortas from hypertensive 2K-1C rats. We have found decreased sensitivity to the NO donor TERPY in the aortic rings from 2K-1C rats compared to normotensive 2K rats. In addition, the reduction in [Ca2+]c in response to TERPY is impaired in VSMCs from 2K-1C rats. In previous studies carried out in our laboratory, we also reported a reduction in the sensitivity to NO in the aortas from 2K-1C rats, although this was done for different NO donors [5,6]. Some authors have attributed this impairment to an increased O 2 production in the aorta from hypertensive rats [10,20,32], since NO reacts with O 2 and forms peroxynitrite (ONOO ), thereby reduc ing NO bioavailability. The major O2 source in VSMCs is the NAD(P)H oxidase, which is stimulated by angiotensin II [33]. It is well recognized that the development and maintenance of Goldblatt hypertension 2K-1C is dependent on the renin–angiotensin system [34]. Rajagoplan et al. (1996) have demonstrated that angiotensin II infusion can cause hypertension and increase in the vascular O 2 production secondary to the activation of NAD(P)H-dependent oxidases. In this way, elevated angiotensin II levels could be activating NAD(P)H oxidase and increasing O 2 in the VSMCs from 2K-1C rats. We used vitamin C as a pharmacological tool to investigate whether the impaired effect to TERPY in the aorta from 2K-1C rats could be due to elevated O 2 concentrations. Our results show that the relaxant effect to TERPY is normalized in the aortic rings from 2K-1C rats incubated with vitamin C (100 lM). In the same way, the decrease in
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[Ca2+]c in response to TERPY was normalized in VSMCs from 2K-1C rats incubated with vitamin C (100 lM). This treatment does not modify the effect to TERPY in the aortas and VSMCs from 2K rats. Therefore, it can be suggested that vitamin C does not modify the NO release from TERPY, but it reduces the O 2 levels in the aortic segments from 2K-1C rats. The free radical-scavenging effect of antioxidant vitamins such as vitamin C is well documented [35]. Also, some studies have shown that the antioxidant properties of vitamin C are associated with the decreased activation of NAD(P)H oxidase and the increased activity of superoxide dismutase, which is an antioxidant enzyme that metabolizes free radicals [20]. In addition, as reported by Newaz et al. (2005) there is an increase in nitric oxide synthase (NOS) activity with addition of dietary vitamin C [36]. In the present study, our results indicate that vitamin C increases the effect of NO on aorta from hypertensive rats. According to Bonaventura et al. (2007), the relaxation induced by TERPY is due to release of radicalar NO (NO ) and nitroxyl anion (NO). Therefore, we should consider the possibility that vitamin C also scavenges the NO. However, our results suggest that it is not the case because the incubation of the aortic rings with vitamin C did not change the effects of TERPY in the isolated aortas and VSMCs from 2K rat aortas. The NO basal levels were measured using a fluorescent NO probe (DAF-2 DA) in the confocal microscope. We verified lower intracellular NO basal levels in the aortic VSMCs from 2K-1C rats. In the same way, the increase in [NO]c in response to TERPY is lower in the aortic VSMCs from 2K-1C rats when compared with 2K rats. Therefore, we could verify that the NO bioavailability is reduced in VSMCs from 2K-1C rats, which could account for the impaired effect to TERPY in the aorta from 2K-1C. It was not possible to measure the [NO] in presence of vitamin C, since it is a quenching agent for DAF-2T reaction product of NO and O2 [31]. Superoxide quantification in the aortic segments was performed by HPLC analysis. The O 2 concentration is higher in the aortas from 2K-1C rats than those from 2K rats. Nevertheless, vitamin C normalizes the O 2 concentration in the aortas from 2K-1C rats. These results agree with those of other studies, which have described increased O 2 concentrations in the aortas from renovascular hypertensive rats [8–10]. In the same way, other authors have shown that the treatment with antioxidants normalizes O 2 concentration in the aortas from hypertensive rats [8,10,20,26]. In conclusion, our results show that the impaired effect to TERPY in the aortas from 2K-1C rats is due to increased O 2 concentration that is normalized by vitamin C. Acknowledgments We thank Ma´rcia C.Z. Graeff for technical assistance in the confocal microscopy (Faculdade de Medicina de Ri-
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